Camellia nitidissima Chi (or name Camellia nitidissima) is a precious and rare plant in the world, which is as famous as Cathaya argyrophylla, Alsophila spinulosa, Davidia involucrate and other precious living plant fossils. Founded in 1993 by Chinese botanist Jinglie Zuo in Fangcheng of Guangxi Province, Camellia nitidissima Chi is one of eight national first-class protective plants and included in plant species in Annex II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. At present, 40 breeds and 5 variants of Camellia nitidissima Chi are well known to the world, mainly distributed in Vietnam and southern edge of subtropical zone and northern edge of tropical zone within the borders of Guangxi Province in China.
Camellia nitidissima Chi, Inula nervosa Wall, Noni Puree, Yeast β-glucan, and Tissue culture of Saussurea involucrata are new resources food approved by the No. 9 document in 2010 of Ministry of Health of China. The leaves and flowers of Camellia nitidissima Chi are enriched in total flavones (isoflavonoids, biflavonoids, anthocyanin and neoflavonoids etc.), tea polyphenols, multiple amino acids and microelement etc. by studies.
As one kind of compounds widely existed in the nature, flavonoids are the derivative of chromane, characterized in that it is with basic framework of C6-C3-C6. Flavonoids can be divided into dozens of categories: flavones, flavanols, isoflavones, flavanones, flavanonols, aurones, flavanones, anthocyanidins, chalcones and chromones etc., over 4000 kinds of flavonoids are found at present, mainly existed in the leaves, fruits, roots and skin of plants, experimental results show it is of extensive physiological and pharmacological activity (including antioxidant, scavenging oxygen free radicals, antivirus, anticancer, anti-inflammatory, anti-aging etc.), so the study on flavonoids has become a hot topic in Chinese and foreign medical profession. Traditional extracting methods mainly include ultrafiltration, enzymolysis, adsorption by coarse pored resin, supercritical fluid extraction, ultrasonic method and microwave field extraction etc. (Rui Zhang, Yaqin Yu, Yang Shi. Study on Flavonoids Extracting Technology. Food and Machinery, 2003, 01, 21-22), while they are always restricted by extracting efficiency or cost.
At present, most studies on active ingredients of flavonoid substances in Camellia nitidissima Chi are confined to traditional ultraviolet and chromatographic detection or solvent extraction, membrane separation etc., like Zhihua Zhan performed qualitative analysis on the chemical component in leaves of Camellia nitidissima Chi. The flavonoids in Camellia nitidissima Chi are occupied about 2.33% by qualitative analysis. (Zhihua Zhan, Extraction and Separation of Flavones in Leaves of Camellia nitidissima Chi[D], Guangxi Normal University, 2006); repeated silica gel column chromatography, Sephadex LH 20 gel chromatography, ODS chromatography and repeated recrystallization and other methods are adopted by Xiao Peng and co-workers to perform separation and purification on chemical component in flowers of Camellia nitidissima Chi, and the structure is identified on the basis of physicochemical constants and spectrum analysis. 13 compounds including flavonoids are obtained from the separation of ethanol extract in flowers of Camellia nitidissima Chi. (Xiao Peng, Dayong Yu, Baomin Feng, Ling Tang, Yongqi Wang, Liying Shi. Study on Chemical Component in Flowers of Camellia nitidissima Chi. Guangxi Plant, 2011, 31(04), 550-553). The content of total saponins, total polyphenol (tannin) and total flavones in different plant parts of Camellia nitidissima Chi, Camellia pubipetala, Camellia impressinervis, Camellia chrysantha and Camellia chrysanthoides is detected through ultraviolet spectrophotometry by Qian Tang. The content of total saponins, total polyphenol(tannin) and total flavones of alcohol extract in flowers of Camellia nitidissima Chi is respectively 21.30%, 6.56% and 21.76%. (Qian Tang, Yanying Luo, Yongqi Wang etc. Quantitative Analysis on Chemical Component of Camellia nitidissima Chi Section, Lishizhen Med Mater Med Res, 2009, 20(4), 769-771); while Quanbin Chen and co-workers take following method, the leaves of Camellia nitidissima Chi are used as the material to extract flavones, high purification aglycone is obtained by hydrolyzing the flavones, solvent extraction, column chromatography and other methods; structure characterization is performed by melting point, TLC, liquid chromatography, infrared spectroscopy, nuclear magnetic resonance method and other analysis methods to confirm the leaves of Camellia nitidissima Chi contain flavonoid glycosides, the aglycon of which is quercetin and kaempferol. (Quanbin Chen, Zhihua Zhan, Qiaoyun Zhang, Na Liao. Separation and Purification of Flavones Aglucone in Leaves of Camellia nitidissima Chi and Characterization. Guangxi Tropical Agriculture, 2005, 6, 10-11).
Flavonoids in Hedyotis diffusa are extracted by microwave-assisted technology by Caixia Zhang and co-workers, and the extraction processes are optimized, the primary and secondary sequence of affecting flavones extraction ratio is as follows on the basis of identification of chemical structure of extract, extracting temperature, ethanol concentration, extracting time and solid to liquid ratio, the best extraction process is ethanol concentration 80%, extraction time of 40 min, solid to liquid ratio of 1:100 under 40° C., the extraction ratio of flavones in Hedyotis diffusa reaches to 2.86% under this condition. (Caixia Zhang, Dingjian Cai, Wenying Fang. Optimization of Flavones Extracting Process and Structure Identification. Anhui Agricultural Sciences, 2011, 39(01), 131-133); Jialin Li and co-workers discussed the effect of extraction temperature, extraction time, solid to liquid ratio and ethanol concentration on the total flavones extraction ratio of Rhus chinensis. The extraction ratio is the best under extraction time of 10 min, extraction temperature of 60° C., solid to liquid ratio of 1:60 and ethanol concentration of 70%. Experimental results show the extraction ratio of total flavones reaches to 6.65% under the best conditions. (Jialin Li, Suzhen Wu, Zhanyu Bei. Study on Extraction Process of Total Flavones in Rhus Chinensis).
Dendrimer is a new class of three-dimensional and highly ordered macromolecules in recent years. PAMAM is one of the most widely and deeply studied dendrimer, with highly branched and highly symmetrical structure and a large number functional groups on the surface and other unique structural features, PAMAM is widely used in drug carrier, surface active agent, catalyst, nano materials, membrane materials and other fields. One generation of obtained product will be increased every time the structural unit of dendrimer is repeated and multiplied, at present, 10.0G divergent method is the leading and most mature synthetic method in the synthesis of PAMAM dendrimer, this method is of mild reaction condition, rapid reaction and high selectivity. (Huimin Tan, Yunjun Luo. Dendritic Polymer [M]. Beijing: Chemistry Industry Press, 2001).
PAMAM dendrimer is of rich amine terminated structure and inner secondary amine and quaternary amine structure, so PAMAM dendrimer presents faintly alkalinity. With phenolic hydroxyl group, most flavonoids present faintly acidity, it is easy to be dissolved into water containing soda, and it can be precipitated out after acidification. The first reason is the acidity of flavone phenolic hydroxyl group, and the second is the ring opening of mother nucleus of flavone under alkaline conditions, so 2′-hydroxychalcone is formed, with the rise of polarity, it is dissolved. (Xuefeng Guo, Yongde Yue. Research Progress of Extraction, Separation and Purification of Flavonoids and Content Measuring Method. Anhui Agricultural Sciences, 2007, 35(26), 8083-8086). According to the present invention, the high efficiency separation of the flavonoid substances in Camellia nitidissima Chi is realized by the extraction and adsorption effect of PAMAM dendrimer with faintly alkalinity and flavonoid substances with faintly acidity.
For better separation of the flavonoid substances adsorbed by PAMAM, the magnetic nanoparticles-PAMAM nano composites of the present invention are prepared by compounding PAMAM and magnetic nanoparticles, like ferrites (Fe3O4, γ-Fe2O3 and MeFe2O4, wherein Me=Co, Ni, Mn), Fe, Co, Ni, alloy particles and iron nitrides (FeN, Fe2N, ε-Fe3N and Fe16N2) and other particles, separating the magnetic nanoparticles-PAMAM nano composites extracted and adsorbed with faintly acidity flavonoid substances from extract by magnetic effect; and finally performing separation on the adsorbed flavonoid substances with magnetic nanoparticles-PAMAM nano composites by ultrasound-assisted extraction.